Self-monitoring of blood glucose is important for individuals with diabetes to be aware of their usual glucose levels and to use them for treatment. Enzymes having glucose substrates are employed in sensors for self-monitoring of blood glucose. One such enzyme is glucose oxidase (EC 1.1.3.4), which has an advantage of being highly specific to glucose and having high heat stability. For this reason, it has been used as an enzyme in blood glucose sensors. The first announcement of such properties goes back to as long as forty years ago. In blood glucose sensors that utilize glucose oxidase, the blood glucose level is measured when electrons generated in the process of converting glucose to D-glucono-d-lactone by oxidization are conducted to an electrode via a mediator. However, glucose oxidase poses a problem in that it tends to transfer protons produced by the reaction to oxygen, causing dissolved oxygen to adversely affect the measured values.
One solution to address this problem is to use a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent glucose dehydrogenase (EC 1.1.1.47) or pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (EC1.1.5.2; formerly EC1.1.99.17) as the enzyme in blood glucose sensors. These enzymes have an advantage of being free from the influence of dissolved oxygen. However, NAD(P)-dependent glucose dehydrogenases have poor stability and are cumbersome, often requiring a coenzyme. Similarly, PQQ-dependent glucose dehydrogenases have poor substrate specificity and react to saccharides other than glucose, such as maltose and lactose, thereby deteriorating the accuracy of the measurement values.
WO Patent Application Publication No. 2004/058958 discloses an Aspergillus-derived, flavin-bound glucose dehydrogenase. Since the activity of this enzyme on xylose is only 10% of that on glucose, in the case of measuring the blood glucose level of an individual taking a xylose tolerance test, the accuracy of the measured value may be impaired. In addition, the enzyme has a residual activity ratio of about 89% after treatment at 50° C. for 15 minutes, thereby exhibiting good heat stability. Moreover, WO Patent Application Publication No. 2006/101239 discloses nucleic acid and amino acid sequences of the enzyme.
U.S. Pat. No. 7,662,600 discloses a modified FAD-GDH having improved heat stability in liquid when compared to an FAD-GDH derived from wild-type FAD-GDH. The modified FAD-GDH is derived from a eukaryote, especially a filamentous fungus such as an Aspergillus spp., and as having a primary structure with at least one amino acid substituted, deleted, inserted or added to FAD-GDH.
US Patent Application Publication No. 2008/220460 discloses a modified FAD-GDH derived from an Aspergillus spp. (e.g., Aspergillus oryzae or Aspergillus terreus) having improved heat stability when compared to wild-type FAD-GDHs. This reference focuses only on a modified FAD-GDH produced by gene recombination in Escherichia coli. Consequently, the FAD-GDHs are non-glycosylated enzyme variants that were screened only under liquid conditions. The reference therefore is silent about specific modifications to the nucleotide sequence to obtain FAD-GDH variants that are glycosylated and that have improved heat stability under dry conditions by eliminating or inactivating a potential glycosylation site.
For some uses of FAD-GDHs, the heat stability under dry conditions is of special importance. For instance, and with respect to test elements for blood-glucose measurements, the enzyme properties of FAD-GDH in dry chemistries needs to be improved.